Department of Pharmacology and Toxicology, Medical College of
Virginia/Virginia Commonwealth University, Richmond, Virginia
Our laboratory demonstrated that morphine exhibits a modulatory control
over the glyburide-binding site (sulfonylurea receptor) of the
ATP-gated K+ channel. This study evaluated the effect of
chronic morphine administration on the sulfonylurea receptor during
tolerance and physical dependence. ICR and Swiss-Webster mice were
rendered tolerant to morphine by pellet implantation and were withdrawn by pellet removal. Alterations in the Bmax
and KD were evaluated in mouse spinal cord
using the radiolabeled ATP-gated K+ channel blocker
glyburide. The ED50 for Swiss-Webster mice shifted from 13 to 451 mg/kg and thus they were more tolerant to morphine than ICR mice
(ED50 shift from 12 to 120 mg/kg). Swiss-Webster mice were
also dependent to morphine only when the morphine pellet was in place,
unlike ICR mice, which were dependent for 48 h after morphine
pellet removal. Glyburide binding increased during chronic morphine
treatment in Swiss-Webster mice by over 2-fold (from 294 to 635 fmol/mg
of protein). This was not observed in ICR mice. In Swiss-Webster mice,
chronic morphine treatment also significantly increased the
KD by 3-fold (from 0.38 to 1.1 nM), whereas
there was no change in affinity for ICR mice. Both strains of mice
remained tolerant for 2 days after spontaneous withdrawal from
morphine. However, the only increases in the
Bmax and KD of
glyburide were observed in Swiss-Webster mice that were highly tolerant
to morphine. These results indicate that a high degree of tolerance is
needed to alter ATP-gated potassium channels.
 |
Introduction |
Opioids
produce analgesia and have been used for thousands of years to treat
acute pain. However, opioids such as morphine are limited in their
function due to the development of tolerance and physical dependence.
The goal of this article is to evaluate the alterations that occur at
the KATP channel during morphine tolerance and
physical dependence to ultimately determine whether openers of
KATP channels could be useful analgesic agents in patients who have undergone morphine therapy. Ocaña et al. (1995)
evaluated the effects of the KATP opener cromakalim
administered (i.c.v.) on the enhancement of the antinociceptive effects
of various opioids, including morphine. They concluded that openers of
the KATP channel enhance the analgesic effects of opiates.
There is no cross-tolerance between morphine and potassium channel
openers or displacement in receptor binding studies, suggesting that
the interaction between opioids and KATP openers is
indirect (Welch and Dunlow, 1993). These data suggest KATP
openers can be used in combination with morphine to reduce the dose of
morphine needed to produce antinociception, thereby preventing morphine
tolerance and dependence. Such studies also indicate that
KATP openers may be useful during morphine tolerance to
produce antinociception.
Morphine produces antinociception by binding to the µ-opioid receptor
and inhibiting cAMP, which decreases cAMP-dependent protein
kinase activation via Gi/o proteins. Decreasing cAMP-dependent protein kinase activation decreases calcium entry via
phosphorylation-sensitive voltage-gated channels and thus contributes
to morphine-induced antinociception (Olson and Welch, 1991; Bernstein
and Welch, 1995). It has been shown that acute morphine administration
opens potassium channels (Werz and MacDonald, 1985; North et al., 1987,
Aronsen, 1992; North, 1992) producing cellular hyperpolarization
resulting in decreased calcium entry (Adamson et al., 1987; Olson and
Welch, 1991). Conversely, chronic morphine administration leads to a decrease in the amount of potassium that leaves the cell. The cell does
not become hyperpolarized, calcium is allowed to enter (Williams et
al., 1982; Werz and MacDonald, 1985; North et al., 1987; Triggle, 1990;
Aronsen, 1992), and neurotransmitters are again released (Lavidis,
1995). This series of cellular events contributes to morphine tolerance.
Openers of potassium channels produce antinociception that is
attenuated by opiate antagonists, suggesting they release endogenous opioids. In 1993 Welch and Dunlow administered the KATP
openers minoxidil, lemakalim, and diazoxide (i.t.), showing they
produced antinociception that was attenuated by the KATP
blocker glyburide, as well as the opiate antagonists naloxone (s.c.)
and ICI 174,864. Glyburide (i.t.) has been shown to produce a
withdrawal syndrome in morphine-tolerant mice (Welch and Dunlow, 1993),
as well as a partial antagonism of morphine-induced antinociception
(Ocaña et al., 1990; Welch and Dunlow, 1993). Raffa and Martinez
(1995) showed that the supraspinal administration of glyburide also
produces a rightward shift in the dose-response curves of morphine and methadone. Studies evaluating the effect of glyburide on other opioid
receptors indicate that glyburide administered (i.c.v.) not only
antagonizes µ-agonists but also the 
-agonist
[D-Pen2,D-Pen5]-enkephalin,
implying KATP channel involvement in the antinociceptive effect of - and µ-opioid receptors (Wild et al., 1991).
The following experiments were undertaken to assess whether morphine
tolerance and dependence alter ATP-gated potassium channels. Using two
strains of mice shown to vary in the metabolism of opioids, studies
were performed to determine 1) how the number of glyburide-binding sites (sulfonylurea receptors) change during morphine tolerance and
dependence; 2) how these receptor changes correlate with the time
course of tolerance and dependence; and 3) what the possible mechanism
underlying the difference in morphine potency between ICR and
Swiss-Webster mice may be. Previous studies in our laboratory have
shown that Swiss-Webster mice chronically treated with morphine show an
increase in the Bmax and
KD of [3H]glyburide
binding in the brain and spinal cord (Welch et al., 1997). The
KD in the brain increased by 167% and the
Bmax increased 63%. In the spinal cord,
the KD increased by 193%, whereas the Bmax increased by 238%. Welch et al.
(1997) evaluated the effect of glyburide during morphine tolerance, but
not physical dependence.
We determined the ED50 values in ICR and
Swiss-Webster mice using morphine and methadone, two well studied
µ-agonists, as well as
morphine-6-
-D-glucuronide (M6G), an active
metabolite of morphine. Methadone was evaluated to determine whether
the strain differences noted with morphine-induced antinociception could be replicated using another µ-agonist of similar potency and
efficacy. Comparison of the ED50 values for M6G
in both mouse strains was done to determine whether the differences
between the strains were due to differences in the metabolism of
morphine to M6G.
| |
Materials and Methods |
Chronic Morphine Treatment.
Male Swiss-Webster and ICR mice
(Harlan Laboratories, Dublin, VA) were implanted with either a 75-mg
morphine pellet or placebo pellet according to the method of Way et al.
(1969). The mice were administered supplemental (s.c.) injections of
either morphine (20 mg/kg) or saline twice per day for 3 days. Animals
treated in this manner were used for either behavioral studies or
binding studies.
Treatment Regimen for Morphine-, Methadone-, and M6G-Treated
Mice.
Antinociception was determined using the tail-flick latency
assay with a 2- to 4-s baseline to "flick" the tail and 10-s
cut-off (D'Amour and Smith, 1941). Dose-response curves were obtained from naïve, morphine-treated, placebo-treated, and
spontaneously withdrawn mice that were previously placebo or morphine
treated. The mice were administered (s.c.) morphine for the
determination of tolerance. In addition, (s.c.) morphine, methadone, or
M6G were tested for antinociception in naïve animals only.
Tail-flick latencies were determined 20 min post administration of the
drugs. %MPE was calculated as follows: (drug time 
control
time/10 control time) × 100.
Binding Studies.
Representative groups of placebo- and
morphine-pelleted mice (not used in any experiments) were tested for
tolerance by administering morphine (20 mg/kg s.c.) and tested for
antinociception 20 min later. The remainder of the treated mice were
considered tolerant if the representative group met the criteria for
tolerance. The lack of antinociception (%MPE <10%) in the
morphine-pelleted mice was the criterion for the development of
tolerance. In the placebo-pelleted mice, the same dose of morphine
produced >95% MPE.
For [3H]glyburide binding studies, animals were
sacrificed and spinal cord tissue was taken from mice that were either
chronically treated, with morphine or placebo pellets intact, or
spontaneously withdrawn by pellet removal. Synaptosomes were prepared
from mouse spinal cord using subcellular fractionation techniques
described by McGovern et al. (1973). Please refer to Welch et al.
(1997) for a full description of the preparation of synaptosomes.
Binding was performed according to the method of Mourre et al. (1989) with slight modifications. Synaptosomes from the spinal cord (0.6 mg/ml
protein; 2-ml final volume) were incubated for 60 min (equilibrium binding was found to be reached at 40 min) at 4°C in 20 mM HEPES/NaOH buffer, pH 7.5, with the concentrations of
[3H]glyburide (50 Ci/mmol, 99% pure) from 1 pM
to 1 µM. Incubations were stopped by rapid filtration through Whatman
GF/B filters soaked for 1 h in polyethylenimine to reduce
nonspecific binding. Filters were washed with 100 mM Tris-HCL, pH 7.5, at 4°C and counted. Nonspecific binding was measured using 0.1 mM glipizide.
Physical Dependence Studies.
Mice were rendered tolerant by
the method described above and once tolerance was ascertained (under
Binding Studies), the placebo and morphine pellets were
removed for 24, 48, or 72 h. The pellets remained intact in other
groups of mice. To test for physical dependence, mice were given
naloxone (1 mg/kg s.c.) and immediately placed on a circular platform
approximately 1.5 ft in height for no more than 15 min (Takemori and
Sprague, 1978). Thus, precipitated withdrawal was ascertained in mice
previously spontaneously withdrawn. "Percent jumped" was calculated
as the number of mice that jumped divided by the total number of mice in the group.
Statistical Analysis.
Significant differences between
treatment groups were determined using the Student's t test
and chi square values. ED50 values and
parallelism were ascertained using the methods of Tallarida and Murray
(1987) for graded dose-response data. All binding assays had an
n 
3 and the results from the separate experiments
were averaged. The Bmax and
KD values were obtained from Scatchard plot
analysis using the LIGAND program (Munson and Rodbard, 1980).
Drugs.
[3H]Glyburide was obtained
from New England Nuclear (Boston, MA); cold glipizide, as well as the
rest of the above-mentioned reagents, were purchased from Sigma
Chemical Co. (St. Louis, MO). Morphine and morphine pellets, as well as
the placebo pellets were obtained from National Institute on Drug Abuse
(Bethesda, MD).
| |
Results |
Morphine Is More Potent in ICR Than Swiss-Webster Mice as Measured
by the Tail-Flick Latency Test.
A stringent protocol for inducing
tolerance was used for this study. The mice were implanted with
morphine pellets as well as injected twice per day with 20 mg/kg
morphine. Both Swiss-Webster and ICR mice chronically treated with
morphine for 72 h were tolerant to morphine as indicated by a
rightward shift in the dose-response curve (Fig.
1, A and B). Basal morphine
antinociception differed between the strains as noted by the difference
in the ED50 of morphine in naïve
Swiss-Webster and ICR mice (Table
1). Chronically treated
Swiss-Webster mice had an ED50 potency ratio of
35.2 compared with placebo and ICR mice had an
ED50 potency ratio of 10.2 compared with
placebo-implanted mice. Swiss-Webster mice were therefore 3.5 times
more tolerant to morphine than ICR mice.
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Fig. 1.
Dose-response curves of s.c. morphine in
naïve ( ), chronic placebo- ( ), and chronic morphine-
( ) implanted Swiss-Webster (A) and ICR (B) mice. The antinociceptive
effects (as %MPE) were measured as described under Materials and
Methods, using the tail-flick latency test with n = 5-8 mice/dose.
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Duration of Tolerance after Spontaneous Withdrawal from Morphine Is
the Same in Both ICR and Swiss-Webster Mice as Measured by the
Tail-Flick Latency Test.
The duration of tolerance was also
evaluated to determine whether there was a correlation between the time
course of tolerance and changes in glyburide binding. Both ICR and
Swiss-Webster mice remained tolerant to morphine for 48 h after
abrupt withdrawal from morphine (Fig. 2,
A and B). There was a time-dependent leftward shift in the
dose-response curves for ICR and Swiss-Webster mice as tolerance began
to wane. The ED50 values for chronic
morphine-treated mice and morphine-treated mice withdrawn for 1 day
were statistically higher than those of their corresponding placebo
treatment groups. The level further decreased after 2 days, but
remained significantly higher than placebo mice with pellets removed
for 2 days. After 3 days of withdrawal, the morphine-treated group did
not differ statistically from the corresponding placebo group (Fig.
2B). These data were consistent with those found in Swiss-Webster mice. Chronically treated Swiss-Webster mice had an
ED50 of 451 mg/kg (401-507) that was
significantly higher than that of the chronic placebo-treated mice.
Although the ED50 of morphine-treated mice decreased after 1 day of spontaneous withdrawal, it was still significantly higher than placebo. The ED50
further decreased in morphine-treated mice after 2 days of spontaneous
withdrawal yet remained higher than the corresponding placebo-implanted
mice. The ED50 value of morphine-treated mice
returned to a level no different from placebo after 3 days of
withdrawal (Fig. 2A).
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Fig. 2.
Dose-response curves of s.c. morphine in mice allowed
to spontaneously withdraw from chronic morphine for 1 ( ), 2 (),
or 3 ( ) days. represents mice implanted with a morphine pellet
and chronically treated with morphine (20 mg/kg s.c.) two times per day
for 72 h. A, results in Swiss-Webster mice. B, results in ICR
mice. The antinociceptive effects (as %MPE) were measured as described
under Materials and Methods, using the tail-flick latency
test with n = 5-8 mice/dose.
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Duration of Physical Dependence after Spontaneous Withdrawal from
Morphine Was Longer in ICR Than Swiss-Webster Mice as Observed in the
Platform-Jumping Test.
The time course of dependence was
determined in both ICR and Swiss-Webster mice to determine whether
there was a correlation with changes in glyburide binding (Fig.
3). Morphine- and placebo-treated mice
were administered naloxone (1 mg/kg s.c.) and immediately placed on a
jumping platform to determine whether they were dependent (jumped) or
nondependent (did not jump) on morphine. ICR mice were dependent during
chronic treatment and after 2 days of spontaneous withdrawal followed
by precipitated withdrawal. During chronic treatment, 100% of the
morphine-treated mice jumped, which was significantly higher than
chronic placebo-treated mice. After 1 day of spontaneous withdrawal
followed by precipitated withdrawal, 88% of the mice jumped, which
again was significantly greater than placebo-treated mice after pellets
were removed for 1 day. Withdrawing the mice for 2 days resulted in
86% of the mice jumping, which remained significantly higher than the
corresponding placebo-treated group. There was an abrupt cessation of
dependence at 3 days of withdrawal. Swiss-Webster mice were physically
dependent on morphine only during chronic treatment with 100% of the
mice jumping from the platform as shown in Fig. 3A.
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Fig. 3.
Platform-jumping test for dependence in
Swiss-Webster and ICR mice. Mice were either chronically treated with
morphine ( ) or placebo-pelleted ( ). They were allowed to
spontaneously withdraw by cessation of treatment and pellet removal for
the indicated days. Mice were administered a 1-mg/kg s.c. dose of
naloxone and placed on the jumping platform for no more than 15 min. A,
results in Swiss Webster mice. B, results in ICR mice. No column
indicates that none of the mice in that group jumped. Withdrawal was
measured as % jumped as described under Materials and
Methods with an n = 5-8 mice/dose.
*P < .05, **P < .01, and
***P < .001.
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|
Chronic Morphine Treatment Increased Both the
KD and Bmax of
[3H]Glyburide Binding to the Sulfonylurea Receptor in
Swiss-Webster, but Not ICR Mice.
There was an increase in the
KD in Swiss-Webster mice chronically
treated with morphine (Fig. 4). The
KD increased almost 3-fold from 0.38 ± 0.024 to 1.1 ± 0.113 nM. The Bmax,
which is the number of glyburide-binding sites, was twice as high in
Swiss-Webster mice chronically treated with morphine versus those that
were placebo-pelleted. The Bmax increased
from 294 ± 31 to 635 ± 117 fmol/mg of protein (Fig.
5). No changes were seen in ICR mice during chronic treatment and neither strain showed changes in Bmax or KD
during spontaneous withdrawal (Figs. 4 and 5). In Swiss-Webster mice,
there is a statistically significant difference in the
KD and Bmax
between chronic placebo- and placebo-treated mice that were
spontaneously withdrawn (Table 2). This
increase was seen on days 1 and 3 and returned to chronically treated
levels by day 10 (Table 2). Mice were exposed to metofane for pellet
implantation and withdrawal. We believe that the increase in
Bmax and KD is stress-induced due to the surgical procedure and possibly to the exposure to the anesthetic agent. There was not as much variability in
the ICR strain. There was no correlation with the time course of
tolerance and changes in the number of glyburide-binding sites for
either strain. However, it appears that the time course of physical
dependence in chronic morphine-treated (morphine pellets intact)
Swiss-Webster mice correlated with alterations in both the
KD and Bmax of
glyburide for the sulfonylurea receptor.
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Fig. 4.
Analysis of the affinity
(1/KD) of [3H]glyburide to
the sulfonylurea receptor in mouse spinal cord in naïve mice
( ), mice chronically treated with either morphine () or placebo
pellets (), and mice allowed to spontaneously withdraw from morphine
for 1, 3, or 10 days. A, results in Swiss-Webster mice. B, results in
ICR mice. #, values significantly different from chronic placebo and
*, values significantly different from corresponding placebo values.
n 3. *P < .05, **P < .01, and ***P < .001.
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Fig. 5.
Analysis of [3H]glyburide binding
(Bmax) to the sulfonylurea receptor in
mouse spinal cord in naïve mice (), mice chronically treated
with either morphine () or placebo pellets (), and mice allowed
to spontaneously withdraw from morphine for 1, 3, or 10 days. A,
results in Swiss-Webster mice. B, results in ICR mice. #, values
significantly different from chronic placebo and *, values
significantly different from corresponding placebo values.
n 3. *P < .05, **P < .01, and ***P < .001.
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Differential Effects of Morphine versus Methadone and M6G in
Swiss-Webster and ICR Mice.
Dose-response curves were determined
in both strains of mice using methadone, a µ-agonist, and M6G, a
metabolite of morphine. The dose-effect curve for both M6G and
methadone produced a full agonist effect. Methadone was more potent
than morphine in producing antinociception. However, there was no
difference in the antinociceptive effects of methadone between the two
strains of mice (ED50 potency ratio of 1.53)
(Fig. 6). M6G produced an antinociceptive
effect that was similar to that of morphine. Like methadone, there was no difference in the antinociceptive effect between the two strains of
mice when M6G was administered (ED50 potency
ratio of 1.64) (Fig. 7). Although the
dose-effect curves for methadone did not differ significantly from
parallel between the two strains of mice, the dose-response curves for
M6G did differ significantly from parallel between the two strains.
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Fig. 6.
Dose-response curves in Swiss-Webster () and ICR
( ) mice after the s.c. administration of methadone. The
antinociceptive effects (as %MPE) were measured as described under
Materials and Methods, using the tail-flick latency test
with n = 5-8 mice/dose.
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Fig. 7.
Dose-response curves in Swiss-Webster () and ICR
() mice after the s.c. administration of
morphine-6--D-glucuronide. The antinociceptive effects
(as %MPE) were measured as described under Materials and
Methods, using the tail-flick latency test with n = 5-8 mice/dose.
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| |
Discussion |
Previous work in our laboratory has shown that morphine tolerance
increases both the Bmax and
KD of [3H]glyburide
in Swiss-Webster mice (Welch et al., 1997). This study investigated the
ability of morphine to alter the number and affinity of
glyburide-binding sites during tolerance and physical dependence. The
goals of this study were to determine whether there was a correlation
between glyburide-binding site changes and morphine tolerance and
dependence. In addition, a second goal of this project was to determine
whether changes in glyburide binding could account for the increase in
morphine tolerance seen in Swiss-Webster mice. Therapists treating
chronic pain patients know from first-hand experience that patients
respond to morphine in various ways. It is possible that these patients
may differ in their ability to metabolize morphine, or the problem may
lie at the level of the opioid receptor. Therefore, aside from our main
objectives, a third goal of this study was to speculate why the two
strains of mice differ with regard to morphine potency. Understanding these differences could be beneficial therapeutically for
morphine-resistant patients. We have demonstrated that Swiss-Webster
mice become more tolerant to morphine than ICR mice with respective
potency ratios of 35.2 and 10.2. Thus, we believe that a high degree of morphine tolerance is necessary to alter ATP-gated potassium channels.
We expected to see an increase in glyburide-binding site number and a
corresponding decrease in affinity during morphine tolerance in both
ICR and Swiss-Webster mice, with this effect gradually returning to
normal during dependence. What we observed instead was an increase in
Bmax and KD
only during chronic treatment and only in Swiss-Webster mice. Both
strains were tolerant to morphine during chronic treatment, as well as
48 h after treatment ceased and morphine pellets were removed. It
appears that changes in glyburide binding correlate better with
dependence. In this study we show that Swiss-Webster mice are only
physically dependent during chronic morphine treatment and that it is
during this time that changes in glyburide binding occur. There tends
to be more variability in the Bmax and
KD in the Swiss-Webster strain of mice.
Results indicate there is a statistically significant difference between chronic placebo-implanted mice and those in which the placebo
pellet was removed for 1 day and 3 days. This indicates that the Swiss
strain is extremely sensitive to stress, which may lead to alterations
in glyburide-binding site number and affinity in placebo-implanted
mice. The difference in KD and
Bmax were statistically different between
placebo groups but were not different from the paired morphine group.
There were also differences in the ED50 values of
placebo-implanted mice. Again, this further implies that Swiss-Webster
mice are sensitive to the surgery, pellet implantation, and pellet
removal. The issue of sensitivity is addressed in these experiments by
using paired comparisons between placebo and morphine-treated mice that
were exposed to the same treatment regimen. Although the differences in
the KD and Bmax
between chronic morphine- and placebo-treated Swiss-Webster mice were
modest, but significant, these data confirm that a high degree of
morphine tolerance is necessary to alter glyburide binding.
Genetics has been well studied with regard to the varying effects of
opioids on different strains of mice. In an early study, strain
differences were attributed to the turnover rate of neurotransmitters (Maas, 1963). Eidelburg et al. (1975) examined four strains of mice,
including ICR and Swiss-Webster. They showed that there was a genetic
difference between strains with regard to locomotor activity,
tolerance, and dependence. Evaluating the brain uptake of
dihydromorphine ruled out blood-brain barrier penetration as the cause
of the strain differences. Many have hypothesized as to why the strains
differ. Muraki and Kato (1985) postulated that polygenes might be
involved in the regulation of the effects of opioids on locomotor
activity. They also found strain differences in morphine-induced
hypothermia and respiration that could not be positively correlated to
variations in [3H]naloxone binding in seven
different brain regions (Muraki and Kato, 1986). Rady et al. (1990)
showed that Swiss-Webster mice metabolize heroin (typically a
µ-agonist) in such a way that it acts at the -opioid receptor;
however, heroin is a µ-agonist in ICR mice. In addition, antisense
mapping of MOR-1 indicates that morphine and M6G produce
antinociceptive effects via distinct receptors that are splice variants
of the MOR-1 gene (Rossi et al., 1995a,b, 1997). These studies showed
that morphine analgesia is antagonized by MOR-1 antisense
oligonucleotides directed against exons 1 and 4, whereas M6G analgesia
is blocked by antisense oligonucleotides directed against exons 2 and 3 as well as exon 4.
We speculate there may be differences in morphine metabolism and/or
µ-receptor number between the two strains of mice. In fact, morphine
is more potent in ICR than Swiss-Webster mice, suggesting that there
may either be fewer µ-opioid receptors in Swiss-Webster mice or
differences in the metabolic pathway of morphine between the two
strains. To determine whether there was a µ-receptor deficit, a
dose-response curve using methadone was generated. Methadone is a
µ-agonist that is slightly more potent than morphine, but metabolized
differently. Methadone is biotransformed in the liver into pyrrolidine
and pyrroline, which are excreted in the urine and bile (Reisine and
Pasternak, 1996). The ED50 of morphine in
naïve Swiss-Webster mice was 23 mg/kg (19-28), which was
statistically higher than that of naïve ICR mice [12 mg/kg
(9-15)]. However, the methadone dose-response curve did not differ
between strains with the ED50 values of
Swiss-Webster and ICR mice being 6.7 mg/kg (5-8) and 4.4 mg/kg (3-6),
respectively. This indicates that the difference in potency between
strains was morphine specific and may not indicate a general
µ-receptor effect. In addition, this may also imply that the strain
difference may be due to metabolism because methadone is metabolized
differently than morphine. However, Yoburn et al. (1989) showed that
opioid binding in the brain is correlated to the potency of opioid
agonists. In this study two strains of Swiss-Webster mice that differ
in their sensitivity to morphine were used. Taconic Farms mice are 2 times more sensitive to morphine than Charles River Laboratories mice.
The more sensitive Taconic Farms mice had more opioid-binding sites in
the brain than did the less sensitive Charles River mice. Binding
studies have been performed in CXBK (µ-receptor deficient) and
Swiss-Webster mice, which indicate morphine is less potent in the
µ-opioid receptor-deficient mice (Duttaroy et al., 1999). Bmax values using
[3H][D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin
for CXBK and Swiss-Webster mice were 99 ± 7 and 171 ± 2, respectively. The comparative ED50 values of
morphine between CXBK and Swiss-Webster mice were 21.2 (16.2-26.3) and 3.2 (2.6-3.8), respectively. In contrast, binding studies comparing Swiss-Webster and ICR mice performed by Eidelberg et al. (1975) using
[3H]naloxone on brain homogenates (minus the
cerebellum) did not show a difference in µ-receptor number.
The major path of morphine metabolism is glucuronidation to form both
active and inactive metabolites. M6G is a major active product of
morphine. The data obtained from M6G showed that there was no
difference in the ED50 of the metabolite in
either strain of mice. Also, the two curves were not parallel,
suggesting there might be a difference in the mechanism of action for
the metabolite between the two strains. There is, however, a difference
in the ED50's between naïve ICR and
Swiss-Webster mice treated with morphine. These data provide indirect
evidence that there is a distinction in the metabolic pathway of the
two strains of mice, which may explain the differences in sensitivity
to morphine. Rossi et al. (1996) tried to distinguish the mechanism of
action between morphine and M6G. They found that there was no
cross-tolerance between the two drugs, indicating they work through
distinct receptors. Alternatively, subtypes of the MOR-1 gene may
predominate in Swiss-Webster versus ICR mice. It is also possible that
similar to heroin, morphine may have a greater affinity for the
-opioid receptor than for the µ-opioid receptor in mice of the
Swiss strain.
In conclusion, the results of this study indicate that mice must be
highly morphine-tolerant to alter glyburide binding. Outside of our
main objective, we speculated that the difference with regard to
morphine potency between the two strains might be attributed to
metabolism or the predominance of one opioid receptor subtype over the
other, depending on the strain. Clinically, the use of ATP-gated
potassium channel ligands as analgesic agents may be an excellent
choice in morphine-tolerant patients because KATP openers
produce antinociception during morphine tolerance. Another important
observation of note is the separation between tolerance and dependence
in the Swiss strain. Many believe that tolerance and dependence are a
somewhat interconnected phenomenon. This study demonstrated that
although the Swiss strain was tolerant to morphine for 2 days, they
were physically dependent only when a large degree of tolerance was present.
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Abstract
Introduction
subunits in rats.
Neurosci Lett
198:
99-102[Medline].
